This research is proposed in response to NIH Notice Number PA-07-320 (Development of assays for high-throughput drug screening). We have found that anti-oxidants acutely inhibit invadopodia formation in cancer cells. Higher levels of ROS are detected in cancer cells, compared to their non-transformed counterparts, and strikingly some of this ROS is localized in invadopodia. ROS generated by the NADPH oxidase system are necessary for invadopodia formation and function. Knockdown of the invadopodia protein Tks5, which is structurally related to p47phox (an NADPH oxidase component in phagocytic cells) reduces total ROS levels in cancer cells. Tks5 facilitates the production of ROS necessary for invadopodia formation, and in turn ROS modulates Tks5 tyrosine phosphorylation in a positive feedback loop. The human genome contains several NADPH oxidase catalytic subunits, called Nox1, Nox2, Nox3, Nox4 and Nox5. In evaluating the expression of the Nox subunits in human cancer cells, we found that Nox4 and Nox1 are most frequently expressed. Furthermore, knockdown of Nox4 or Nox1 inhibits invadopodia formation and invasive behavior. These data lead us to hypothesize that NADPH oxidases represent novel therapeutic targets for the treatment of disseminated cancers. Testing this hypothesis will be best accomplished using small molecule inhibitors, yet none of available inhibitors have the appropriate selectivity and pharmaceutical properties. We propose to generate Nox selective inhibitors to allow in vivo studies on the role of Nox in cancer progression. We will: establish and validate cell-based assays for Nox activity, as well as specificity assays; conduct a high throughput screen and verify hits; and improve potency and selectivity, in order to generate tool compounds for hypothesis testing in vitro and in vivo. The significance of this research lies in the possibility of identifying a new avenue for therapeutic development. The immediate impact of this research is that it represents an important and necessary first step in the in vivo validation of a new therapeutic target. PUBLIC HEALTH RELEVANCE: The growth of disseminated cancers leads to morbidity and mortality, and there is an urgent unmet need for new therapeutic targets. This research seeks to develop antagonists of an enzyme that mediates invasive behavior of cancer cells, and thus is of relevance to the public health problem of metastatic cancer.